Epidermal Desmosomes Connect Stratification and Inflammation through Actin Remodeling - Project Summary The evolution of cadherins enabled terrestrial metazoans to develop complex tissue barriers, with desmosomal cadherins anchoring elastic and tough intermediate filaments (IF) to the plasma membrane at cell-cell junctions, thus providing mechanical strength to tissues and organs. Uniquely expressed in stratified epithelial keratinocytes, the desmosomal cadherin, Desmoglein 1 (Dsg1), also has more recently appreciated functions in driving the process of stratification through cell delamination and suppressing inflammatory signaling. Surprisingly, these Dsg1-dependent functions may be mediated by shared molecular machinery comprising Arp2/3-associated actin remodeling proteins. Previous studies from my lab demonstrated that Dsg1 recruits an Arp2/3 complex to promote actin polymerization at desmosomes, facilitating keratinocyte migration to the next superficial layer by altering cell mechanics in the stratifying cell. However, the mechanism by which Dsg1 maintains Arp2/3 at the plasma membrane remains unclear. My data points to a role for Nck1, an adaptor protein known to facilitate Arp2/3 activation via N-WASP, which was identified as a potential desmosome interactor in a publicly available proteomic screen. Our data suggest that Dsg1 recruits Nck1 to desmosomes through Dsg1 Y1042, enabling interactions with N-WASP and Arp2/3 at the plasma membrane. Silencing Nck1 or introducing a Y1042F mutation in Dsg1 impairs epidermal stratification, suggesting that Dsg1 regulates actin remodeling via N-WASP through tyrosine phosphorylation at the Nck1 binding site. Notably, the Nck1 associated protein, N-WASP is also present in the nucleus, where it can suppress the expression of inflammatory cytokines, including IL-23, known to cause inflammation in Dsg1-deficient patients and in the common inflammatory disorder psoriasis. These data lead to my hypothesis that through Nck1, Dsg1 recruits N-WASP/Arp2/3 complex in a non-receptor tyrosine-kinase (NRTK) dependent fashion to control stratification and, through downstream signaling, directs N-WASP to the nucleus to help manage inflammation. In Aim 1 I will determine how Dsg1 recruits the N-WASP-actin complex to the membrane to drive actin remodeling for cell delamination using assays to assess protein-protein interactions, actin polymerization, and keratinocyte stratification. In Aim 2 I will elucidate how Dsg1 governs N-WASP localization and stability through serine/threonine kinase signaling to modulate inflammatory pathways using live cell imaging, cell fractionation and analysis of keratinocyte cytokine expression. These studies promise to reveal how Dsg1 integrates its roles in creating the physical and immune barriers through shared mechanisms and may reveal new therapeutic avenues for strengthening and repairing the epidermal barrier.
